Bioclumica et Biophysica Acta, 1137(1992) 127-134

127

© 1992ElsevierScience PublishersB.V. All fights reserved0167-4889/92/$05.00

Secretagogue-induced proteolysis of lung spectrin in alveolar epithelial type II cells U n - J i n P. Z i m m e r m a n

~, D a v i d W . S p e i c h e r ~ a n d A r o n B. F i s h e r ~

"~Institme for Enz.ironmental Medicine, Unit:e."sily of Pennsylcania Medical School, Philadelphia, PA (USA) and b Wtstar institut~ University of Pennsylcania, Philadelphia. PA (USA)

(Received21 November1991) (Revisedmanuscriptreceived25 May 1992)

Key words: Calpain; Alveolar epithelial type Ii cell; Spectrin

lncalbation of isolated rat alveolar epithelial type 11 cells with secretagogues(calcium ionophore, ATP or terbutaline) resulted in rapid proteolysis of lung spcetrin and appe~rance of multiple proteolytic products which showed immunoreaetivity with an antibody against human erythrocyte spectrin. These proteolytic products were similar to those generatexl from erythrocyte spcetrin or cultured lung tumor cells (A549 cells) incubated with purified calpain. Furthermore, incubation of alveolar type I! cells with a calpain-specffie inhibitor modulated the secretagogue-induced pro~eolysis of lung spectrin. Thus, stimulation of secretion appeared to activate endogenous calpain in type II cells, suggestingthat calpain-mediated proteolysis of a submembranous cytoskeletal protein could play an important role in the secretory process.

Introduction The pulmonary alveolus is lined with two phenotypically different epithelial cells, type I and 11 [1]. The cuboidal type !1 cell is the site of lung surfactant synthesis. The suffactant is stored in lamellar bodies and secreted from type I! cells by exocytosis of lamellar bodies [2,3,1. Previous studies have shown that ~cretory activity of type II ~!!s can be incres~eG by various pharmacological stimulL Calcium ionophore, pnrinergic and ~-adrenergie agunists stimulate secretion from isolated alveolar type II cells [2]. The precise pathway by which each secretagogue enhances secretory activity of type II cells has not been clearly defined, but increased int~ceUular calcium is generally involved. In the present study, we have explored the possibility that endogenous cytosolic protease, calpain, may be activated when the intraceliular calcium level is raised as a result of the stimulation of type li cells by various secretagogues. Calpalns (EC 3.4.22.17, calcium-

CorreSl~mdenceto: U.-J.P.Zimmerman,Institute for Envirmtmental Medicine, I John MorganBuilding,Universityof PennsylvaniaMedical School,361hand HamiltonWalk, Philadelphia,PA 19104,USA. Abbreviations:SDS,sodium-.imJecylsulfate;EGTA, ethyleocglycolbi~i~-aminoethylether)N, N, iV', N'-telraacetic acid; EDTA, eghylenediaminetetraacticacid; DTE, dlthioeffthritol; Hepes, N-2-hydrox~t hylpiperazine-N'-2-ethannsalrunicacid.

activated neutral proteases) are found in x~rtually all mammalian tissues and are noticeably abundant in lung [4]. There are two forms of calpain, u-calpain and m-calpain, that differ markedly in levels of calcium required for activation. Both enzymes however have similar substrate specificities [5]. Calpains are characterized by the ability to carry out selective and limite~ proteolytic modification of specific proteins in the presence of calcium. However, the cellular functions of calpains in most tissues have not been identified. Nevertheless, their ubiquitous distribution argues for a fuudamental role for these enzymes in calcium-dependent phenome,na that occur at the cytosol and plasma membranes. Previous studies have indicated that the membrane-associated cytoskeletal proteins appear to be the specific target of calpaln activities. For exaraple, calpains are involved in cytoskeletal alterati, ms associated with activation of platelets [6], secretion of dense granules from neutrophils [7], fusion of myoblasts [8] and fusion of eryt fux)cytes [9]. The present study examines the possible role of calpain in the secretion of surfactant and explores the hypothesis that fusion of secretory vesicles with plasma membrane may be dependent upon proteolysis of membraneons components of alveolar type 11 cells. Hence, we have examined the effects of known secretarogues on the activation of calpain in type II cells.

128 Materials and Methods Calcium ionophore (A23187), leupeptin and dimetbylsulfoxide were purchased from Sigma, St. Louis, MO. Calcium ionopborc we~ solobilized in dimethylsolfoxide. Minimum Essential Mediam (MEM) was purchased from Flow Laboratory. Mclean, VA. Nitrocellulose membrane (0.45 /zm) was purchased from Schleicher and Schuell, Keene, NH. ELISA-grade affinity-.purified horseradish peroxidase-conjugated goat anti-rabbit lgG (H + L) was purchased from BioRad, Richmond, CA. C.alpain inh~itor !! (N-acetylleucyl-ieucyl-methio~-tal) was purchased from Boehringer Mannheim, Indianol~lis, IN, and soiubii~cd in dimethyiso!foxide. Radioactive casein ([t4C]casein)was purchased from N E N / D u p o n t Researcll Products, Boston. MA.

were washed three times with MEM and the cells were cquii~rated mrtber with fresh MEM (2 ml) for 30 min before experiments. Experimental incubations were terminated at pre-dercrmined times by addition of excess EGTA to the incubation medium and immediately cooling on ice. The cells were rapidly washed with three changes of PBS containing 10 mM EGTA or 10 wM EGTA plus leupeptin (100/tg/ml), and harvested by gentle sct'alx'ng. Cells were pelleted and lysed with 100-200 p l of HEED buffer (10 mM Hepes, I mM EDTA, 1 mM EGTA, 1 mM DTE, pH 7.4) containing 0.1% Triton. The iysate and particulate fractions were analyzed for the proteolytic products of lung spectrin by SDS PAGE and Western blot. Under these conditions, the proteolytic products of lung spectrin in the unstimulated control cells were negligible.

Anti-a spectrin Preparation of calpain Calpain was purified from bovine erythrocytes as previously described [10].

Erythrocyte spectrin Human erythroc3"te spectrin was prepared from fresh normal donor blood, as previously described [I 1].

Polyclonal antibodies were raised in rabbits against the monoclonal immunoaffinity-purified 86-kDa segment of a subunit of human erythr~cyte spectrin. Monospecific anti-a-spectrin i m m a n ~ o b u l i n was purified utilizing an a-spectrin-conjugated immunoaffinity column [14].

Western blot A549 cells The human lung adenocarcinoma cell line (A549) obtained from American Type Culture Collection was routinely cultured in MEM supplemented with glutamine (2 raM), sodium bicarbonate (23 mM) and antibiotics (streptomycin and penicillin, 100 U / I each). Stock cultures were passaged at 4 - 5 day intervals. For most experiments, cells were cultured at a monolayer density of about 5-106 cells per T-75 flask and harvested by gentle scraping Harvested cells were washed three times with phosphate-buffered saline (PBS).

Following SDS polyacqdamide gel electrophoresis (PAGE) according to Laemmli [13] using a 7.5% gel, proteins were transferred to nitrocellulose membranes, which were transiently stained with Ponseau red. The NC membrane was then immunoblottod with a rabbit polyclonal antibody to the 80-kDa segment of the a subanit of human erythrocyte spectrin. The antibody was used at a dilution of 1 : 100, the blots probed with horseradish peroxidase conjugated goat anti-rabbit lgG at a dilution of 1 : 1000 and visualized by reaction with 4-chloro- l-naphthol.

Isolated alveolar epithelial type I! cells

Prateolytic activity o f alveolar type H cells

Alveolar type il cells were isolated from rat lung, as previously described by Dobbs et al. [12]. The lungs of an anesthetized rat was cleared of blood in situ, removed from the thorax, lavaged and incubated with elastase. Tissue was minced and filtered through progressively smaller Nitex gauze. Cells were then 'panned' on bacteriologic plastic petri dishes pre-coated with rat IgG to remove macrophages. The supernatant containing unattached cells was centrifuged. The cells were resnspended in Dulhecco's modified Eagle's medium containing 10% fetal calf serum and approx. 5 - 1 0 ° cells were plated for each Falcon Primaria culture dish (35 ram). Isolated cells were greater than 85% type !I by fluorescent staining with phosphine 3R. After 18 h of culture in a i r / C O 2 (19:1), the medium containing unattached cells was discarded. Remaining type II cells ( 1 - 2 - 1 0 ° cells; 30% plating efficiency)

Aliqnots (100/~g) of the cytosolic fraction of type II cells were incubated with [t4Ckasein and calcium (5 mM) for 30 min. The incubation was terminated by addition of bovine serum albumin (0.01%) and trichloruacetic acid (TCA, 10%). Caseinol.~e activity was expressed by the TCA-soluble radioactivity of the sample. Results

Endogenous spectrin in the isolated alveolar type II cells In order to determine the existence of a spectrin-like protein in rat alveolar type II cells, isolated type II cells were extracted with a low-ionic-strength medium, as used for extraction of spectrin from erythrocytes. The low-salt extract was then subjected to SDS PAGE and Western blot using a polydonal antibody to the a

129 subunit of human erythroc3"te spectrin. The immunobiot revealed a 280-kDa band strongly immunostained by the antibody, indicating that endogenous spectrin (lung spectrin) was indeed present in type 11 cells and cross-reacted to erythrocyte spectrin (Fig. 1). in additioi:, three other bands (approx. 190, 170 and 150-kDa), with much less intensity, were also recognized by the antibody. These bands probably represent in-vivo degradation products of lung spectrin, as discussed below.

-280 -190 -170 -150

Calcium ionophore-mediated degradation of endogenous spectrin in alveolar type H cells After 18 h culture, 1.5 - 10° type II cells attached to the culture plate were incubated with calcium ionophore A23187 (0.I t t M ) for 1, 5, 10, 30 and 120 min. Incubations were terminated by addition of excess EGTA and cooling on ice. The control cells had no additions and were processed in'.anediately in the presence of EGTA (10 raM) and leupeptin (100/zg/ml), as

-

90

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80

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60

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54

i

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-280

1

2

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Fig. 2. Calcium ionophorc-mediated degradation of endogenous spectrin in alveolar type !I cells. Isolated type !I cells (1.5- lOb/plnte) were inc~,~ated with calcium ionophoreA23187 (0.1 p.M) for the

indicated iengths of ti.'r~e. The control had no addition and was processed immediately. Aliqunts (120 p.g) e.¢ pellet fraction were analyzed for spectrin degradation products by SDS PAGE (7.5%) and immunoblottedwith anti-a spcetrin. Lanes:(1) 0 rain (control), (2) i min;(3) 5 min;(4) 10min;(5) 30 min;(6) 120min.

Fig. 1. Western bier of low salt extract of alveolar type II cell. holated type It cellswerelysedand exltractedwithlow-ionic-strength medium(0.1 mM EDTA,0.5 mM DTE,0.03 mM phenylmethylsulfonyi fluoride., pH 8.0). A 50-~g aliqum of the low-saltextract was subjected to 7.5% SDS PAGE and immunoblnttedwith polyclonal antibody to the 80-kDa segmentof a subunitof humanetythmcyte spectdn.

described in Materials and Methods. Cells were analyzed for spectrin degradation products by SDS PAGE and immunoblot with anti-a spectrin (Fig. 2). The immunoblot revealed a set of breakdown product bands which included a 150-kDa band with increasing intensity with time. At a longer incubation (120 min), the 190 and 170-kDa higher-molecular-weight product bands had disappeared but the 150-kDa band remained and additional low-molecular-weight bands (90, 60 and 54 kDa) were generated (Fig. 2, lane 6). When the iysate was similarly analyzed for the proteolytic products of lung spectrin, the immunoblot shmved that the same set of 190-, 170, and 150-kDa polypeptides was released into the cytosol. However, more of the 190- and 170-kDa products than of the 150-kDa were present in the lysate at any given time. The results indicate that the 190- and 170-kDa polypeptides were

130 released early into the cytosol while the 150-kDa polypeptide remained largely associated *~th the plasma membrane (data not shown).

Secretagogue-induced proteolysis of lung specttin in alveolar type H cells P.xogenous ATP or terbutaline has been shown to enhance secretion from alveolar type II cells [2]. We have examined the effect of these secretagogues on proteolysis of lung spectrin in type 11 cells. Type ll cells (1.5 - 106) attached to the culture plate were incubated with either A T P (100/zM) or terbutaline (10 /zM) or without any secretagogue, for 10 min at 3"FC. At the end of incubation, cells were processed as descn'bed in Msterials and Methods and analyzed for proteolytic products of lung spectrin. The immunoblot revealed that the distinctive 150-kDa proteolytic product was present in cells exposed to ATP or terbutaline and only faintly in the control cells (Fig. 3). The results

-280

-150

indicate that extracellular ATP or terbutaline can induce in vivo proteol~is of lung spectrin in type II cells.

Proteolytic pattern of erythrocyte or lung spectrin generated by erogenous calpain In order to determine that in-vivo proteolysis of lung spectrin in type 11 cells exposed to secretagogues was due to the catalytic activity of endogenous calpain, we first examined the time-course for formation of proteolytic products from e~jthrocyte spectrin. Purified erythrocyte spectrin was digested with purified erythrncyte calpain in the presence of calcium. Aliquots were withdrawn as a function of time and analyzed by SDS PAGE and stained with C.omnassie blue (Fig. 4A). The results indicated a nearly complete logs of both sp~ctrin subunits within 30 mk'l. The time-course indicated that the a subunit (280-kDa) was cleaved first and multiple proteolyt~ products with mo!e~ular masses of 190, 170, 150, 110, 90 and 80-kDa were sequentially generated. We then compared the proteolytic pattern of lung spectrin generated in situ by purified calpain using human lung carcinoma cells (A549) (Fig. 413). The A549 cell line can provide a homogenous cell preparation without contamination with other cell types or extraneous proteases and shows some of the differentiated characteristics of type I! cells [15]. The immunoblot showed a set of proteolytic products with molecular masses of lg0, 170, 150, li0, 90 and 80 kDa that were vet)" similar to those obtained from erythrocyte spcetrin and type !! cells. In each case, the variations in the intensity of these product bands over time indicated that the 190- and 170-kDa product bands werc earl), proteolytic intermediates which disappeared with prolonged incubation. In contrast, the 150*kDa product was initially absent (Fig. 413, lane 1) but increased in intensity and remained as a prominent product band. Furthermore, the 150-kDa product was specifically absent when the incubation was carried out in the presence of leupeptin (IG0/zM), a thiol protease inhibitor (Fig. 4B, lane 7). Therefore, the 150-kDa polypepfide is a stable proteolytic product of lung spectrin generated by calpain.

Modulation of in-vivo proteolysis of lung spectrin in alveolar t~lTeH cells by calpain-speciflc inhibitor

1

2

3

Fig. 3. Sccretagogue-induceddc~"~d~fionof lung s~ctrin in type 11 cells. Isolated type il cells (i.5-106/plate) in 2 ml MEN were ino.Jbatedwith either ATP (100 tzM) or terbu~aline(10 ttM) for I0 rain. Samples (100 ~g) were analyzed by 7.5% SDS PAGE and immunobk~ttedwith anti-*.-spnetrin.Lanes:(1) ATP; (2) tethutaline; (3) control.

In order to confirm that the secretagogue-indu' ~ d proteolysis of lung spectrin was, indeed, due to tlt~ activity of endogenous calpain in type ii cells, isolated type !! cells were preincubated with cell-permeable m*calpain-specific inhibitor ( I 0 0 / z g / m l medium) for J - 3 h. Calcium ionophore was then added and cells were in,.-ubated further for 10 min to induce proteolysis of lung spectrin. The immunoblot and deasitometric tracings indicated that the intensity of the 150-kDa product band

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-190 -170 -150

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67

Fig.4. Pro~eoty~of et'~ttmcyte spectrinor lungspectrinin-sanby e3a~genouscalpain.(A) Etythrocytespectrin.Humanegythmc~espnctrin(150 pg) dig~',ed -.vithbovineet3~mk-'yle~a.'i~. (10 pg) in the presenceof calcium(3 mM). Prior to calciumaddition, an aliquotwas renmvedfor the initial poinLThe sampleswereanalyzed by7.5% SDS PAGEand stained withCoomassieblue. Lanes:(1) 25/tg sp¢ctrin,0 s; (2) 5 g (3) 301;; (4) ; win;(5) 10 win;(6) 30 win.(B) Lungspectrinin A549cells. A549cellswere h~q~otonicallyI~ed and the cell pellet (450/~g)resuspendedin HEED heifer, was digested withcalpain GOFI0 in the presenceof calc/,rn (3 mM).Alicluotswerewithdrawnas a functionof time..A separate ag~quot(?S/~f,)waspre-incubatedwithleupeptin{100/tM) for 10rain priorto addition of calciumand incubatedfurtherfor 60 win.Aiiquo~(75 t.tg)were analyzedby SDS PAGEand immunoblonedwithanti-a fOcetrin.Lanes:(1) init;al point;(2) 5 s; (3) I win;(4) 5 win; (5) 30 win;(6) 60 win;and (7) 60 rainwith leupeptin.

was substantially reduced by (47%) in cells preincubated with inhibitor II as opposed to cells exposed to calcium innophore alone (Fig. 5A and 13). In order m establish that the reduction in the 150-kDa proteolytie product was due to the depressed calpain activity by the inhibitor, the cytosolic fractions were assayed for calcium-dependent caseino]ytie activities. The result indicated that caseiuoly~ activity was similarly depressed by 54% (n = 2) in cells p~incubated with calpain inhibitor il (Fig. 5 0 . The inability of the inhibitor I! to completely suppress proteolysis may result from a slow rate of cell penetration by the inhibitor and reversibility of inhibition rather than the effects due to non-calpain proteases. The results of the study thus indicate that the 150-kDa band is a calpainspecific proteolytie product of lung spectrin in type !! cells,

Discussion Spectrin-like polypeptides are known to be present in many cell types [16,17]. The importance of specttin in the protein network underlining the inner surface of erythrocyte plasma membrane for membrane stability and for anchoring integral membrane proteins has been well documented [18,19]. Involvement of spectrin in the release mechanism of secretory vesicles has also been suggested [20,21]. Previous studies have shown that crythrocyte or brain spectrins (fodrin) were highly susceptible to calpain-mediated fragmentation [22,23] and that the a subnnit of fedrin was preferentially cleaved by calpaln at the calmodulin binding site [24,25]. In the prese,t study, we have determined that a spectrin-like protein is present in the isolated alveolar type I1 cells and that the lung spectrin in situ was rapidly prote.

132

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1;;0 150-

o.~oo B

0.40o_

o.ooo

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.~

2000

1500

~

1000

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(1)

(2)

Fig. 5. Modulation of calcium-ionophore-ioduced proteolysis of lung spectrio by calpain inha~itor II. Isolated ~ !! cells were lYreincubatedwith cell-permeable culpaio-si~Afic inh:'bitor ( 100 #g/ml medium) for 3 h. Pmteolysis was then induced by addition of A23187 (I #M) and incubated further for 10 mio. The control incubation had no addition. (A) Western blot. Aliquot (80/.tg) was analyzed for lung Sl~Ctrio breakdown products by 7.5% SDS PAGE and imutuitoblotted with anti-a spectrin. Lanes: (1) cells exposed to A23187; (2) cells preineubatod with calpain inhibitor and then exposed to A23187; (3) the control cells. (B) Densitometric tracings of the immunobloL Left panel: cells incubated with A23187. Right panel: cells preineubated with calpaio inh:'bitor and then incubated with A23187. (C) Caseinolyfic activity. C~osolic fraction (50/~g} of the cells was incubated with [t4Ckaseiu and calcium (3 raM) for 30 mio and TCA-soluble radioactivity was counted. Lanes: (i) cells exposed to A2318"/; (2) cells preincnbated with inh~itor and then e~r-~.d to A23187. olyzed by e n d o g e n o u s c a l p a i n in alveolar type II cells s t i m u l a t e d by s e c r e t a g o g u e s . W e have also shown t h a t the h i g h e r m o l e c u l a r w e i g h t p r o d u c t s (the 190- a n d

170-kDa b a n d s ) o f l u n g spectriu w e r e g e n e r a t e d d u r i n g the initial r a p i d p h a s e o f proteolysis. T h e s e early interm e d i a t e s w e r e f o u n d even in the protease-inhibited

133 incubations with leupeptin or calpain inhibitor !1. Their presence attests to the rapidity of proteolysis relative to the rate of inhibition by the inhibitors. O n the other hand the 150-kDa band was the stable proteol)tic product of lung spectrin as the previous studies on erythrocyte spectrin or fodrin have indicated [25-27]. Lung tissue has been known to contain mainly mcalpain (high-calcium-requiting enzyme form) as demonstrated by its enzym~tic activity and m R N A levels [28]. Furthermore, earlier immunohistochemical studies indicated that alveolar type I! e e ~ were primarily stained by anti-m-calpain antibody [29]. in the present study, we found that calpaln-specific degradation of lung spectrin was induced by all three secretagogues, calcium ionophore, A T P or terbutaiine. In the presence o f calcium ionophore, cellular calcium has been estimated to reach as much as 1 m M [31,32]. Therefore, the observed intraceilular cleavage of spectrin could be due to the activation of endogenous m-calpain in type !1 cells. However, the same proteolytic products were also found in type !! cell~ exposed to exogenous ATP, an agonist known to induce only a transient ( 2 - 5 min) rise in cytosolic calcium level (10 p.M) [33]. Hence, o u r results suggest that m-ealpain was also activated a t the / t M intracellulaJr calcium level. Mechanisms by which m-calpain c~ouid b e activated in vivo are not known. Howevey, instances of in vivo activation o f m-calpain have been pr,.~ciously reported, (e.g., stimulation of neutrophiL¢, with phorbol ester-induced proteolysis o f protein kina.~ C by the endogenous m-calpain [34]). m-Calpain .,~olubilized a nuclear protein kinase a t micromolar caL'iom concentrations [35]. Microinjection of m-calpain promoted mitosis in synchronized PtK cells [36]. Terbutaline, on the o t h e r hand, is not known to elevate cytosolic calcium [32,33], but stimulates secretion in association with activation o f c A M P - d e p e n d e n t kinase [37]. Hence, phosphorylation may be a mechanism by which terbutaline could induce activation o f calpain. The phosphorylation at specific sites on or n e a r the m e m b r a n e would serve to sequester calcium and provide a locally concentrated calcium pool. In conclusion, o u r present study indicates that calpain may function in stimulus transduction and may have a role in regulation o f suffactant secretion by cleaving lung spectrin. Secretagogue-induced elevation of second messengers (calcium or c A M P ) activates calpain and subsequent focal dissolution of the submembraneous spectrin network may facilitate closer apnsition and fusion of secretory vesicles with the plasma membrane for exocytosis. Aekmmledgemeats This study was supported by Grant-in-Aid from the American Heart Association (UJZ), Research G r a n t

A w a r d from the A m e r i c a n Lung A s s o c i a t i o n / American Thoracic Society (UJZ) and N I H grants H L 19737 (ABF) and H L 38794 (DWS). W e would also like to thank Dr. Avinash C h a n d e r for many helpful discussions.

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